Production of 1, 4-pregnadienes by bacterial fermentation



atent U 3,037,912 Patented June 5, 1962 3,037,912 PRODUCTIGN F1,4-PREGNADIENES BY BAC- TERIAL FERMENTATION Louis I. Feldman, SpringValley, N.Y., Neil E. Rigler, Ridgewood, N..l., and Anthony J. Shay andBarbara E. Nielsen, Pearl River, N.Y., assignors to American CyanarnidCompany, New York, N.Y., a corporation of Maine No Drawing. Filed May 6,1960, Ser. No. 27,246 8 Claims. (Cl. 195-51) This invention relates tothe dehydrogenation of steroids. More particularly, it is concerned witha novel method of dehydrogenating certain A -steroids of the pregneneseries by means of microbiological fermentation, whereby a double bondis introduced in the 1,2- position.

A number of steroids of the pregnadiene series, such asl-dehydrohydrocortisone, for example, are becoming increasinglyimportant either as therapeutic agents or as intermediates in thepreparation of other therapeutically useful steroids. Such compounds,which are obtained by the practice of the present invention, are usefulas antifiammatory agents in the treatment of arthritis,

asthma, burns, bursitis, and the like, and also in the treatment of skindisorders and collagen diseases. As such these compounds are used incombination with fillers, excipients, etc., in tablets, powders, pills,etc. They canalso be used parenterally in a solution or in a suspension.I

In accordance with the present invention, it has been found that9a-fluoro-1,4-pregnadiene-11 8,l6a,17a-triol- 3,20-dione or itslo-acetate can be prepared by the use of microbiological fermentation toaccomplish the desired dehydrogenation in ring A of such steroids.Suitable bacterial agents for the present process include Bacteriumhavanieizsis and Bacterium mycoides. The reaction obtained in theprocess of the present invention can be illustrated, for example, in thefollowing equation:

in which R is hydrogen or a lower alkanoyl radical and R is oxygen orhydrogen and hydroxyl.

In carrying out the process of the present invention, the organism iscultivated aerobically in a suitable nutrient medium with a A -steroidof the pregnene series. During the growth of the organism underfavorable conditions, two hydrogen atoms are eliminated from steroidring A and a double bond i thereby obtained in the 1,2- position. Theexact mechanism of this dehydrogenation is not wholly certain. It isknown to be caused by en- Zymes produced by the organism in the processof the growth.

A suitable nutrient medium contains a soluble source acids; variousnatural products containing carbohydrates, such as corn steep liquor,soybean meal, cottonseed meal and many other available materials whichhave been used heretofore as a source of carbon in fermentationprocesses. Usually a variety of the above can be employed in the mediumwith good results.

Suitable sources of nitrogen may include, for example, some of theabove-named materials, such as corn steep liquor, soybean meal,cottonseed meal and the like. Various other substances may be utilized,as for example, beef extract, casein, yeast, enzymatically digestedproteins, and degradation products, including peptones, amino acids, andmany other available proteinaceous materials which have been found to besuitable in supporting the growth of bacteria. Other organic andinorganic sources of nitrogen, including urea, ammonium salts, nitrates,and the like, also may be used in the medium as a source of assimilablenitrogen to provide a favorable growth medium for the organism.

Mineral requirements of fermentation are usually supplied in the crudematerials which are often used as sources of carbon and nitrogen oroccur in the water available for use in the process. However, it isusually advisable to supplement the minerals normally present with addedamounts to obtain an optimal growth. Cations and anions which may bedesirable in added amounts include sodium, potassium, calcium,magnesium, phosphate, sulphate, chloride, cobalt, manganese, and variousothers. It is often desirable, also to provide such trace elements asboron, copper, cobalt, molybdenum, chromium and the like.

Growth of the organism takes place under aerobic conditions and suitableaeration, in flasks, for example, can be achieved by agitation on areciprocating or rot-ary shaker or in bottles or tanks by forcingsterile air through the fermentation mixture. In good practice, sterileair should be available in the medium in a ratio to the medium in therange of from about 1:3 to about 2:1 volumes per minute. Agitation inbottles or fermenter tanks is provided by a mechanical impeller. Whilethe organism will grow at temperatures of 5. and 45 C., it is preferablefor optimum results to carry out the process of the present inventionwithin a somewhat more limited temperature range of from about 25 toabout 37 C.

To prepare inocula, 1.0 ml. of washed vegetative cell suspension of theorganism is used to inoculate ml. of sterile medium in a 500 ml.Erlenmeyer flask. An illustrative medium of this type contains thefollowing: 1% cerelose, 0.1% yeast extract (difcc), 0.4% of peptone(Bacto), sodium chloride 0.25% and beef extract (Armour) 0.4%. Thismixture is sterilized for 15 minutes at a temperature of C. (15 poundssteam pressure), and adjusted to about pH 7. This medium is used in theillustrative examples below. The inoculated flask is incubated at 37 C.on a shaker for about 4 to 8 hours. Such inocula may be used toinoculate larger batches of sterile medium in bottles, and such bottlescultures, after fermentation, may be used to inoculate large batches ofmedium infermentater tanks. This procedure is given as a typicalillustration only, and may be varied if necessary or desirable. Forexample, instead of the broth described above, other media may be used.This will be shown in the examples hereinafter.

Typical A -steroids of the pregnene series which can be usefullyprocessed according to the present invention include for example9a-fluoro-4-pregnene-l1 [3-16oc-17ottriol-3,20-dione andl6a-acetoxy-9a-fluoro-4-pregnenc- 11fl,17u-diO1-3 ,2 0-dione.

The amount of steroid added as substrate to the fermentation may bevaried as necessary or desirable. How- $13, 13. of about 0.05 to 1.0gram per liter of nutrient medium.

When using such steroid substrates in the fermentatio r1, ;the productsforme d are the free steroids. These ste oi s-.ate, en ally add d. t thf n i a in. 5 in:

tion or -tin finely dividedh form. preferred method is -di solvethe t ie ha al 9 ot r wa r mi ib s so vents and. add it to; he ermen at ed mtth desired stage in the process. Although the I steroid rnay P qd iqi'fmfl .9l 0nJK 1-Q dtiti .di 'n t n gerierahipr'actiee; jam ss ofpresen out. OfmL-bzitfchs ofinoculatedmediuniiare placed in 100 shakertubes assimilate ;ssasnygr r a iasmcthanol," dlniethyl iforrnarnide', ormixture: thereof. 7

t t as clescribed hereinafter.

This solution is usedforcharacterization of's tejroid conn large'slc'aleffermentations, H v A products'finay be recovered from the,fermentation beer y pl solvent extraction," using a suitablewater-imnii's lil 'solikeng sueh as chlorinated lower hydrocarbons,

be illustrated b thei renewing Zprocedure'l ledpract eml lt i a y be bad on t e q s same trough, being developed simultaneously with thesteroid standard strip. This use of the trough permits the simultaneousdevelopment of many strips. After proper development of the paperstrips, they are removed from theapparatus and; airdried. After drying,the strips'are placed between "a source of ultraviolet light andazincisilicatefcoatedplate, Steroids containing the \*-3;l etoneconjugated! system are observed as dark spots; Strips are 1 linedu pJwith at least one standard strip and Rf,- determined..,-The differentsteroids can be then identified by their positions on the strips.

The desired A -st'eroids will be more polar than their corresponding A-steroid; -It'lshould beunderstocd, more- V over that the desiredA-steroids, once they have been 'isolatedand characterized, maythemselvesbe used in a standardister oid solution for process improvement. 7 f

.I The present inv'iention will be more' fuliy described: in conjunctionwith the following examples. 'Theyfare tended as illustrations;

. "Z'EXAMPLEI: frrpar'mi of; b -rig re-l,4grigaaaieae-l1, 315 7a;.

tube agarslairt ofBizeferizim liavgniansis (ATCC No; 4001).is'waslied'with 7 ofsterile saline solution, and the resultingsuspension isused'to inoculate 100ml;

of sterile' me'dium (described 'hereinbefore) in a 509ml.

' Erlenmeyer'flaskfllhe mixture is incubated on a 'recipro eatingshaker'for about 7 hours'at 37 C. One pertions of this culture 'are'then used to inoculate forty-eight I 100 ml. lots of sterile mediumiirSOG m1. flasks and the inoeulatedflasks incubated for about 16 hours,at about 28o.C..: ,1 p v ;At this time mg'.-of 9ot-flu0ro-4-pregnene-118,160:- l7bc-triol-3,20-dione' is dissolved in 2.0 ml. of methanol and'added to each flask." The incubation is then continuedfor an'additional72 hours. A '5' ml. aliquot is extracted once'with 15 ml. of ethylacetate and the extract is "ev ap'oratecljto'drynessunder vacuum." Thedry residue is taken up in a methanol-dimethylformamide mixture and anappropriate sample analyzed bythe paper str'ip-techthe crass rogram s:

alcohols, esters,ketoris, etc. Further 'purific atiori' and separationof steroid products from extracts may be accomplishedflbyrnthodswelhunderstodd by those skilled in-theartb Separation} andpurification o'fi asteroidmixture often requirsthe use of Chromatographyf Tlii*proccss employeddoidentify thefste'roidspr'esent in"the'extracted fermentation beerpreviously described is f by paper stripchromatography. fA solvent system used isflpetroleunrether'zbenzenmacetic acidzp-dioxane prepared'byshaking thesolvents 'listedinthe proportheflowei' layeris placedin anop'en dish on-the' fioo'r of aaarge gtass cylinder; The upper layer isthe' solvent phaseand is "usedto fill the trough-shaped well 'within'the '5 1 respectively" in aseparatoryffunnelfand ingthetwo layersftoseparate. A portion ofcylinder; Foreom arison, astanda'rd steroidsolution'is prepared bydissolving a sample ofster'oidj in methanol, di'rhethyl forr'nainide',or mixtures thereof. At least onc standard steroidsolution 5 ischromatographed simultaneously each time an unknown solution is tested.

Exactly 01010 of the standard steroid test solution is "applied: to {thepaper strip atfthe starting line; four inches" from the upper end of thestrip," which: is folded over the edge "of 'the trough and jimm'ersedjinfthe ,solye'nt janotherstripyqiurnif off ine unl inown soluti'on is V's'inlilarly'applidandthis' 'strip is thenfolded'into the EXAMPLE 2.

Repeating the procedure used in Example 1, but substitutinglEhtcterz'l-nn mycoides (ATCC No. 4004) for that used therein, producedthe same result as shown by chromatographic assay.

I EXAMPLE 3 Isolation from Fermentation M ashv I Four liters of whole asproduced in Example 1 are extracted with two 4 liter-portions of ethylacetate, the 'extractsarepo'oled and thecombined ethyl acetatesolutionis concentrated under vacuum t0 dryness. The dry residue is washed withabout 4 ml. of ethyl acetate and a few ml. of ether to ObtairiofiO mg.of light brownish solid. This was chromatographed using an equilibratedsolventsys'tem comprising water, cyclohexane and'dioxane (ratios 114:5parts) and the major portion, as shown'by ultraviolet analysis, isconcentrated under reduced pres sureto produce a crystalline productwhich is' collected, washed severaltimes withdmlfportions ofacetone,'and dried, yielding 613mg. The product, after recrystallizationfrom acetone-petroleum ether, has the followin g properties: k max.=239mu; e=15,600; ['a] =+51; M.P;:275 5279" C. which was not depressed whenmixed with an authentic'sample of 9a-fluo'ro-l,4-pregnadiene-"11,6,l6o;;17e-triol+3,20-dione from'another source. The infraredspectrum the same as thatof the authentic -sample.

EXAMPLE 4 Preparation of 16a-Acet0xy-9a-Flu0r0-11B-1 70c- Dihydroxy-I ,4-Pregnadiene-3 ,20-D ione A portion of 100 mg. of9a-fluoro-11B-16a-17a-trihydroxy-l,4-pregnadiene-3,20-dione is mixedwith 2 ml. of pyridine and 1 ml. of acetic anhydride and allowed tostand over night at room temperature. The so-reacted mixture is dilutedwith water and the product solids are filtered off, washed with Waterand dried. Crystallization of these solids from an ethylacetate-Skellysolve B mixture gave 16a-acetoXy-9 oc-flIlOI'O-l1fi-17a-dihydroxy-1,4-pregnadiene-3,20-dione as needles (75 mg), MP.242244.

Analysis.Calculated for C H O F; C, 65.70; H, 7.65. Found: C, 65.69; H,7.17.

We claim:

1. A process of dehydrogenating one of the group consisting of9a-fluoro-4-pregnene-11fi,16a-17a-triol-3,20- dione and thecorresponding 16a-acetoxy compound which comprises the step ofsubjecting the steroid to the fermentative action of one of Bacteriumhavaniensis and Bacterium mycoides.

2. A process which comprises the step of subjecting one of the groupconsisting of 9a-fiuoro-4-pregnene-11,6,16a, 17atriol-3,20-dione and thecorresponding l6a-acetoxy compound to the fermentative action ofBacterium mycoides and recovering therefrom the corresponding 1,4-

\pregnadiene.

3. A process for the production of 1,4-pregnadienes which comprisescontacting in an aqueous medium under submerged fermentation conditions,one of the group consisting of9a-fluoro-4-pregnene-11fi,16ot,17u-triol-3,20- dione and thecorresponding 16a-acet0xy compound with the dehydrogenating activity ofBacterium havaniensis.

4. A process which comprises: inoculating a nutrient medium containingassimilable carbon, nitrogen and mineral salts with one of Bacteriumhavaniensis and Bacterium mycoiaes adding one of the group consisting ofafiuoro-4-pregnene-11,8,16a,'17a-triol-3,20-dione and the correspondingISa-acetoxy compound continuing the resultant fermentative action on thesteroid until a substantial amount of corresponding A -steroid of thepregnadiene series has been produced, and recovering said producttherefrom.

5. A process according to claim 4 using Bacterium havaniensis, ATCC No.4001.

6. A process according to claim 5 in which the substrate steroid is oneof 9a-fluoro4-pregnene-11fi,16u,17u- -triol-3,20-dione and thecorresponding 16a-acetoxy compound and the recovered product is9a-fiuoro-l,4-pregnadiene-l lfi,1604, 17a-triol-3,20-dione.

7. A process according to claim 4 using Bacterium mycoides, ATCC No.4004.

8. A process according to claim 7 in which the substrate steroid is oneof 9ot-flu0ro-4-pregnene-1'1p,16ot,17ottriol-3,20-dione and thecorresponding 16u-acetoXy compound and the recovered product is9a-fluoro-1,4-pregnadiene-l15,1604,17a-triol-3,20-dione.

References Cited in the file of this patent UNITED STATES PATENTS2,793,164 Fried et al May 21, 1957 2,844,513 Wettstein et a1 July 22,1958 3,016,335 Stoudt Jan. 9, 1962 OTHER REFERENCES Prescott et al.:Industrial Microbiology, McGraw-Hill Book Co., Inc., 1959, pages 725,726 and 749.

Bergeys Manual, 6th ed., Williams and Wilkins Co., 1948, pages 915 and918.

Bergeys Manual, 7th ed., Williams and Wilkins 00., 1957, page 1018.

1. A PROCESS OF DEHYDROGENATING ONE OF THE GROUP CONSISTING OF9A-FLUORO-4-PREGNENE-11B,16A-17A-TRIOL-3,20DIONE AND THE CORRESPONDING16A-ACETOXY COMPOUND WHICH COMPRISES THE STEP OF SUBJECTING THE STEROIDTO THE FERMENTATIVE ACTION OF ONE OF BACTERIUM HAVANIENSIS AND BACTERIUMMYCOIDES.